Regulation of dihydrofolate reductase and other folate-requiring enzymes

.REGULATION

OF

REDUCTASE

AND

DIHYDROFOLATE OTHER

REQUIRING

FOLATE-

ENZYMES*

J. R. BERTINO~" and B. L. HILLCOAT* Departments of Pharmacology and Medicine, Yale University School of Medicine, New Haven, Connecticut

INTRODUCTION

DURING the past 20 years, the investigations of several laboratories have led to the understanding of the important role of the coenzyme forms of folic acid in cellular metabolism, especially in nucleic acid biosynthesis (reviewed ~'n 1-4). Many of the en.zymes utilizing the folate coenzymes have been purified extensively from bacterial and mammalian sources, and one, 10-formyl tetrahydrofolate (FH4)** synthetase, has been obtained in crystalline form from Clostridium cylindrosporum and

Clostridium acidiurici (5). Despite this rapid increase of our knowledge of folate metabolism, relatively little information has been forthcoming that deals with the regulation of the activities of the enzymes involved. This paper will attempt to summarize the status of these s~udies with particular emphasis on the enzyme FH2 reductase in mammalian tissues. I. E N Z Y M E S O F " " O N E

CARBON" TRANSFER

lO-Formyl FH, synthetase..Several bacterial species utilizing purines for growth (5,6) have high levels of 10-formyrFH4 synthetase. Rabinowitz (7) has postulated that the function of the enzyme in these instances is to generate ATP via the reverse reaction (equation 1),** FH4+ A T P + HCOOH ~ 10-formyl F H 4 + A D P + Pi

(1)

*This research was supported by Grants CA-08010, CA-02817 and CA-08341 from the U.S. Public Health Service. tCareer Development Awardee of the National Cancer Institute. ~.lnternational Post Doctoral Fellow (2-FO-5 TW-984-03). **The following abbreviations are used: FH4, tetrahydrofolate; FH2, dihydrofolate; MTX, methotrexate; IMP, inosine monophosphate; AICAR, 5-amino-4-imidazole carboxamide ribotide. 335

336

J.

R. B E R T I N O

AND

B. L.

HILLCOAT

since I 0-formyl FH4 may be generated from purine c a t a b o l i s m - (equation I M P + FH4 ~ 10-formyl F H 4 + AICAR.

(2)

In other bacterial species, this enzyme activity is low or not detectable (6,8), but is present in almost all mammalian tissues investigated(9). Every guinea pig organ assayed (10), and erythrocytes and leukocytes of several species (11) contained 10-formyl FH4 synthetase activity. Reticulocytes contain higher levels than mature erythrocytes(1 l, 12); higher levels were also found in acute leukemia leukocytes and in leukocytes from patients with chronic granulocytic leukemia as compared to normal(12,13). The importance of this enzyme in cell metabolism has not been clearly established; 10-formyl FH4 can also be generated from the oxidation of 5,10-methylene FH4 via the enzyme 5,10-methylene FH4 dehydrogenase, another enzyme with a widespread distribution. The finding that 10-formyl FH4 synthetase activity is higher in immature cells than In differentiated cells, together with the report by Albrecht and Hutchinson(8) that'the level of this enzyme in a MTX resistant strain of Streptococcus faecalis is high and is repressed by the addition of a purine (adenine, guanine or hypoxanthine) but not a pyrimidine (thymine or uracil), would indicate that this enzyme can play an essential role in purine biosynthesis. Another compound which appears to exert a regulatory effect on this enzyme in Micrococcus aerogenes is formate, which induced enzyme-synthesis when added to the growth medium (6). The relationship of this enzyme activity to the growth cycle has been studied in Streptococcus thermophilus and S. faecalis(14, 15). In S. thermophilus, the enzyme activity" increased in the beginning of the lag phase and reached a maximum early in the log phase of growth. Two separate maxima were found during the growth of S. faecalis, the first in the middle of log phase and the second at the beginning of the plateau stage of growth. Studies of the level of this enzyme activity during growth of synchronized mammalian cells are in progress in this laboratory. The regulation, if any, of this enzyme activity in mammalian tissues has not yet been elucidated. One possibility that has been investigated is a possible feedback effect of purines on this enzyme; however, little or no effect was demonstrable when several purines were tested(8,16). Another observation of interest is the report by Lansford et al.(17), who found that spermine, and to a lesser extent, polyamines, stimulated the rate of formyl-FH4 formation catalyzed by this enzyme prepared from Lactobacillus arabinosus 17-5 and also Lactobacillus casei. These organisms show growth stimulation by spermine when the supply of "one carbon" units in the growth medium is limiting. This stimulation resulted from the enhancement of binding of FH4 to the enzyme. The significance of this

REGULATION OF DIHYDROFOLATE REDUCTASE

337

observation is not clear since this stimulatory effect has not been shown to be specific; monovalent cations can also stimulate this enzyme activity. 5,10-Methylene FI-I4 dehydrogenase. This enzyme reversibly interconverts the folate coenzymes 5,10-methylene FH4 and 5,10-methenyl FH4 (equation 3) 5,10-methylene FH4+ NADP ~ 5,10-methenyl FH4+ NADPH + H + (3) Like 10-formyl FH4 synthetase, this enzyme activity was found to be elevated in immature cells (reticulocytes, acute leukemia, bone marrow cells) when compared to the more differentiated forms of these cells (11, 12). In Salmonella typhimurium (18) and a mutant6f E. coil K12, auxotrophic for either methionine or vitamin Bn(19), purine ribonucletide triphosphates were found to inhibit 5,10-methylene FH4 dehydrogenase in vitro. ATP, GTP, and ITP at concentrations of 10-aM inhibited the enzyme competitively with respect to NADP +, while mononucleotides or nucleosides were less active. The high concentrations of triphosphates necessary for inhibition, and the competitive nature of this inhibition with NADP +, also noted for other dehydrogenases, makes the relevance of these observations to in rive control mechanisms less certain. Some evidence for regulation of the rate of synthesis of this enzyme was obtained in both the auxotrophic E. coil K12 strain (met- or B~.:) (19-21) and purine auxotrophs of S. typhimurium(18). Growth of the mutant of E. coil Kn in media containing high concentrations of purines led to partial repression of NS, Nl°-methylene FH4 dehydrogenase. Further evidence for the control of the level of this enzyme activity by purine nucleotides was afforded by the observation that when endogenous pools of purine nucleotides were depleted by growing a purine auxotroph of S. typhimurium in growth-limiting concentrations of adenine, or in adenosine 3'-phosphate, which is-poorly transported, enzyme derepression was observed (18). 5,IO-Methylene FH4 reductase. This important enzyme has been identified and studied only during the last few years (21-24). The formation of 5-methyl FH4 catalyzed by this enzymet (equation 4) appears to be an essentially irreversible process (21). 5,10-methylene FH4+ FADH2 ~ 5-methyl FH4+ FAD

(4)

This step, and the subsequent enzymatic conversion 0f homocysteine to methionine, are reactions of great interest in that they should be under t i n rat liver, N A D P H has been found to be a much better electron donor than N A D H or FADH2 (24).

338

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B E R T I N O A N D B. L. H I L L C O A T

strict metabolic regulation, since this is the only enzyme described that can regenerate FH4 from 5-methyl FH4 (equation 5). 5-methyl FH4 + homocysteine

B~2coenzyme

) FH4 + methionine

S-Adenosyl-methionine

(5)

The requirement for a B12 coenzyme in some systems and also for Sadenosyl-methionine in catalytic amounts is also of interest. In a mutant of E. coli K12, auxotrophic for'either 1-m.ethionine or vitalnin B~2, 5,10methylene FH4 reductase was found to be repressed by the addition of high concentrations of methionine to the media while synthesis of the enzyme was increased by low levels of methionine (18). A study of this enzyme from rat livers by Kutzbach and Stokstad(25) ind]caf~d that 5,10-methylene FH4 reductase activity increased when the diet was free of methionine; furthermore, this enzyme activity was inhibited 90% by 10-3M S-adenosyl-methionine. This inhibition has been f o u n d t o be "allosteric" in nature (15). These studies, if confirmed, provide evidence for a regulatory role of S-adenosyl-methionine in vivo. 5-Methyl FH4-homocysteine transmethylase. In several species of bacteria, both a vitamin B12 dependent system utilizing 5-me-thyl FH4 and a vitamin B~2 independent system, utilizing the triglutamate of 5methyl FH4 have been discovered(26, 27). When cobalamin was added to the growth media, the vitamin Ba2 independent pathway was repressed (27). FH4 also inhibited the cobalamin independent synthesis of methionine from serine and homocysteine: this inhibition was found to be due to a competitive interaction between the folates (FH4 and the triglutamate of FH4) for 5,10-methylene F H4 reductase (28). This inhibition may play a role in regulating methionine biosynthesis; thus the vitamin B12 independent pathway would be inhibited when present, and vice versa. Wang et al. recently reported that two folate dependent methionine synthetizing pathways are present also in rat liver(29). Of additional interest was the distribution of these enzyme activities in the cell fractions (29). The vitamin B~2 independent system, requiring 5-methyl tetrahydropteroyltriglutamate, was found in highest concentration in the mitochondrial fraction, while the vitamin B~2-dependent enzyme activity, utilizing FH4, was found primarily in the cytoplasmic fraction. This is the first evidence that a vitamin B~2 independent pathway utilizing a folate cofactor also exists in mammalian tissues and that it may be located in the mitochondria. Further extension of these studies will be awaited with interest. Serine-transhydroxymethylase. This enzyme (equation 6) catalyzes the interconversion of serine and glycine; it has been detected in bacteria (30), plants (31), and in animal tissues (32). l-serine + FH4 ~ glycine + 5,10-methylene FH4.

(6)

REGULATION OF DIHYDROFOLATE REDUCTASE

339

Unlike the other folate enzymes, except possibly for the previously mentioned Bl~-independent methyltetrahydropteroyltriglutamate-homocysteine transferase, this enzyme activity is associated primarily with the mitochondrial fraction of the cell (29, 33). A possible regulatory role for 5-methyl FH4 and 5-formyl FH4 has been suggested by Schirch and Ropp(34), based on the observation that both these compounds are potent competitive inhibitors of FH4 in the enzymatic conversion of serine to glycine. Glycine was found to have a greater affinity for the enzyme-FH4 complex than for the enzyme alone. This was also the case for the enzyme-5-formyl FH4 and enzyme-5methyl FH4 complexes. Thus, the result of an increase in the level of these inhibitory FH4 compounds in the cell might act to inhibit the serine to glycine conversion.

I1. D I H Y D R O F O L A T E

REDUCTASE

Distribution and characterization. During the past several years, this enzyme has been extensively purified from bacteria(35-37), mouse tumors(38,39), calf thymus(40) and chicken liver(4t,42). The dual role of this enzyme to catalyze not only the formation of FH4 from folate, but also to participate in thymidylate biosynthesis is shown in equation 7. 5,10-Methylene FH4 + deoxyuridylate.

"FH2 + thymidylate.

Ak

+ "Cf' |

FH4<

FH2 reductase NADPH

I

(7)

In this latter capacity, this enzyme serves to reduce the FH2 formed in the formation of thymidylate, thus regenerating FH4. The "C1" unit may be donated by serine or other compounds containing a potential one carbon unit. Surveys of the level of FH2 reductase activity in tissues of various mammalian tissues have been made(10,43,44). In the rat, guinea pig and rabbit, the enzyme was readily measurable in liver, kidney, spleen and intestinal mucosa. Rabbit spleen showed the highest content of enzyme in one study(44). Muscle, lung, heart and brain contained low levels of this enzyme activity in most species examined(44), and tissues from the dog, cat, Rhesus monkey and man were low in enzyme activity compared to rodents (44). The function of this enzyme in liver, an organ with low mitotic activity, may relate more to a role in the production of reduced folates for storage or transport to other tissues (presumably as 5-methyl FH4) than to its role in thymidylate biosynthesis. The difference between the low levels of FH2 reductase in leukocytes or erythrocytes compared to the high levels in bone marrow cells or I2

340

J. R. B E R T I N O A N D B. L. H I L L C O A T

cells from patients with chronic myelocytic or acute leukemia is even more striking than noted for the other folate enzymes (12,45-47). Thus, a decrease in this enzyme activity accompanies differentiation oferythrocytes and leukocytes. This point is emphasized since an understanding of events leading to inactivation of this enzyme activity during normal differentiation may be important in understanding the "induction" of FH2 reductase by MTX discussed in the next section. The folate antagonists, methotrexate (MTX, amethopterin), aminopterin and dichloromethotrexate, are all potent inhibitors ofFH~ reductase, and are valuable drugs in the chemotherapy of malignant disease. The development of resistance to these agents has been studied in bacteria (48), cultured cells, and in routine and human leukemia cells. Several biochemical mechanisms of resistance have been demonstrated: high enzyme mutants (49-51) defective transport mutants (52,53) and mutants having an altered FH~ reductase enzyme with a decreased affinity for MTX(54). High enzyme mutants for murine leukemia cells resistant to MTX have been developed that have an increase in FH2 reductase activity 10-300 over the parent line. Comparison of the properties of the enzymes from the mutant and wild strain cells showed no differences(55-57), indicating that the increased activity observed is most likely due to the presence of an increased amount of enzyme protein. This could result from, (1) increased synthesis, (2) decreased degradation, or (3) both. The factor(s) causing this increase in enzyme activity have not yet been resolved. In man, an increase in FH2 reductase activity has been noted to accompany MTX treatment in both normal and leukemic leukocytes, and in erythrocytes within a few days after methotrexate therapy was initiated(58-59). In this circumstance, since selection of stable mutants with a high level of enzyme seemed unlikely, this phenomenon was termed "induction", to indicate that an increase in enzyme activity occurred, without implying any mechanism. Once it was found that single, non toxic doses of MTX(10-20 mg) could cause a measurable increase in red cell and leukocyte FH2 reductase activity, the kinetics of this "induction" could be established(5859). Thus, as shown in Table 1, the rise occurred within hours as measured in lysates of normal bone marrow cells, or of the peripheral blood cells of patients with acute leukemia. On the other hand, only after 4-5 days was the rise noted in normal peripheral leukocytes or erythrocytes. These findings, together with the demonstration that the concentration of MTX in these cells closely paralleled the rise and fall of the enzyme activity, led to the hypothesis that the increase in FH~ reductase seen occurred during the early stages of cell development, and that in normal

REGULATION OF DIHYDROFOLATE REDUCTASE

341

TABLE 1 Increase in Dihydrofolate Reductase in Human Blood Cells After MTX Administration Cell type Normal leukocytes(6)* Erythrocytes (4) Bone marrow leukocytes(2) Chronic myelocytic leukemia leukocytes(4) Acute lymphoblastic leukemia leukocytes(3) Acute mycloblastic leukemia leukocytes(3)

Time to initial increase

Time to peak rise

5 days 4 days

9 days 6 days

6-24 hr

not determined

24 hr

2-6 days

6-24 hr

not determined

6-24 hr

not determined

Patients received a single 20 mg dose of MTX intravenously. Dihydrofolate reductase activity was measured at pH 8.5 as described (59). *Indicates number of patients studied. subjects, the slower increase in the e n z y m e activity of peripheral blood cells corresponded to the time necessary for these cells to pass from the marrow out to the peripheral blood(58,59). T h e lag phase was appreciably shorter when the activity was measured in bone marrow cells or in immature leukemic leukocytes, since no or little transit time was involved. T h e peak value of the activity measured is thought to indicate the time required for the majority of cells to appear in the peripheral blood after they are labeled with M T X " i n d u c e d " enzyme. T h e subsequent decrease in activity is thought to represent the removal of these cells from the circulation. T h e s e findings form the basis of a rhethod for the study of leukocyte kinetics: either tritium-labeled M T X within cells or the "induction" of FH2 reductase activity caused by M T X may be followed as a leukocyte "tag" (60, 61) . . . . . Specificity and prevention of enzyme induction. Attempts to induce FH2 reductase activity by naturally occurring folate compounds have so far been unsuccessful. Neither folic acid nor 5-formyl FH4 treatment caused a rise in this e n z y m e activity in human subjects(58) or in the dog, a species in which M T X "induction" of FH2 reductase in leukocytes was also observed. Additionally, FH~, FH4 and 5-methyl FH4 did not result in a rise of this e n z y m e activity in the dog(59). On the other hand, potent inhibitors of FH2 reductase other than M T X such as aminopterin, dichloromethotrexate, and pyrimethamine (2,4-diamino-5-p-chlorophenyl-6-ethyl pyrimidine), caused an increase in the e n z y m e activity in dog leukocyte extracts(59). This rise was shown to be specific for F H z reductase inhibitors, since no e n z y m e rise occurred in normal or leukemic leukocytes of patients treated with other chemotherapeutic drugs (64).

342

J.

R. B E R T I N O

AND

B.

L. H I L L C O A T

A search for naturally occurring feedback inhibitors of this enzyme activity(62,63), or for compounds that might prevent the "induction" of dihydrofolate reductase in vivo, has not been rewarding. Thus, in the dog, administration of reduced forms of folate, thy.nidine or inosine, did not block the MTX-induced rise in leukocyte FH2 reductase(59). In man, concomitant actinomycin D administration with M T X was also unsuccessful in preventing the rise in enzyme activity(59). As pointed out previously, these studies cannot be considered to be conclusive, since a negative effect may relate to one of several possible factors that are difficult to control in the whole animal.

Measurement of total and "free" enzyme-The importance of pH. In the studies outlined thus far, an increase in FH2 reductase was noted despite the presence of M T X in the cell extract. Two questions arose: (I) was the total enzyme activity even higher, and (2) what was the actual amount of "free" enzyme activity present in the cell, and how did it relate to the in vitro measurement of enzyme activity? The binding of MTX to FHz reductase, although exceedingly "tight"(65), was found to be reversible and less "tight" at an alkaline pH (66). Indeed, the reason that the rise was observed was that the assay conditions used (pH 8.5 with FH2 as substrate), together with dilution of the cell extract, allowed dissociation of the enzyme and inhibitor. After dialysis at an alkaline pH in the presence of a high concentration of salt or by gel filtration of Sephadex with the same conditions, enzyme extracts could be almost entirely freed of bound MTX, and total enzyme activity could be measured (59). The activity of extracts treated in this manner was considerably greater than that measured without prior separation of the enzyme-MTX complex; this difference (i.e. between "free" and total enzyme activity) was even more striking when the enzyme activity was measured at pH 6.0 before and after separation of the MTX from the enzyme. This difference in binding of 2,4-diamino inhibitors to the enzyme as a function of pH has been ascribed to the ability of the pronated species of the inhibitor to bind more tightly to the enzyme (66-68).

Comparison of the "induced" erythrocyte and leukocyte enzyme with "non-induced" bone marrow enzyme. Since the increase in enzyme activity noted after M T X treatment might be due not only to an increase quantitatively but also to the formation of a different protein, with, for example, a higher maximum velocity at a similar concentration of FH2, a comparative study of "induced" and "non-induced" enzyme has been initiated. Inasmuch as FHz reductase activity present in normal leukocytes or erythrocytes is very low, normal bone marrow cells were used as the source of "induced" enzyme. Enzyme from "induced" leukocyte and erythrocyte FH2 reductase was partly purified and separated completely from MTX. The "induced" enzymes had the following properties similar

REGULATION OF DIHYDROFOLATE REDUCTASE

343

to those of the enzyme partly purified from bone marrow; pH optima, inhibition by MTX, molecular weight, and cation activation(69). Increase o f F H 2 reductase activity in cultured mammalian cells after exposure to M T X (70). During the logarithmic stage of growth of lymphoblast-like cells of human origin (RPMI 4265) in suspension culture, the FH2 reductase activity of the cell extracts increased 3-fold and fell to a basal level as the cells entered the stationary phase of growth (Fig. 1). If the cells were grown in the presence of MTX at a concentration (10 -s M) which did not alter either cell growth or the rate of incorporation of uridine or thymidine into DNA, this pattern was altered. The results of such studies showed that, while the enzymic activity measured at pH 7.0 was not changed, the enzyme activity measured at pH 8.5 Showed a 2-fold increase (Fig. 1), and total ("free" plus bound) enzymic A =

'~

1,0

M

r

% 0.8

~.

aM

15

o

x ~.

w

_~

~o

-> 0.4

~ J u

g

5

~ 0.2

50

I00

150

200

HOURS

Fro. 1 Variation of FH, reductase activityin cells grownin the absence and presence of MTX. To one series of cells (M), MTX (1 × 10-8 M) was added,but not to the other (C). The activityshownfor the M series has not been correctedfor inhibition of enzyme activityby MTX present in extracts (from Hillcoat et al. (70), reprintedby permissionof the NationalAcademyof Sciencepress). activity increased 12-fold during the log phase of growth. The subsequent decrease of enzymic activity to the low levels found when the untreated cells entered the resting phase did not occur in MTX treated cells (Fig. 1). The increase in FH2 reductase activity depended on the time during cell growth at which MTX was added (Fig. 2): The maximum effect of M T X was observed in log phase growth; during late resting phase, the drug produced no effect. Cycloheximide did not prevent the MTX dependent increase of enzyme activity, indicating that protein synthesis was not necessary for such an increase.

344

J. R. BERTINO AND B. L. HILLCOAT A

= - /

CONTROL

65

0.8 0.4

- - 0 . 8

{

~_o K

0.4

O.B[ ?,\

96

R4

*

0.8 o~

" --

04

0.4

u ~ .J

/

u

0.8t

,~ ~

48

0.8 .

'

'

a'6

'

~

,~o

"x'l--L-=

'

ao

0.4

,~o

HOURS

F=6.2 Effect on FH2 reductase activity when M T X was added at periods during cell growth. Arrows indicate the time in hours at which MTX was added to produce a concentration of 4 × 10-8 M. The activity shown is that actually measured at pH 8.5 and. has not been corrected for inhibition of enzyme activity by MTX when present in the extracts. The added M T X did not alter the growth rate of the cells in any case.o--o, cell count: x---x, enzyme activity at pH 8.5 (from Hillcoat et al. (70), reprinted by permission of the N ational Academy of Science press). DISCUSSION

As the tight binding of the MTX to FH2 reductase is known to protect the enzyme from proteolysis(71,72) and from heat denaturation(38), stabilization of the enzyme against normal intracellular degradation seems the most likely interpretation of our experimental observations. As MTX is a competitive inhibitor of FH2, and binds to the enzyme at the site of this natural substrate, the enzyme-stabilizing effect of the inhibitor in vivo may represent an exaggeration of the protection afforded the enzyme by its natural substrate. Thus, the increase of enzyme activity with MTX represents a type of "cofactor induction"(73). Indeed, the failure, in all but one instance, to demonstrate end-production inhibition, induction (increased rate of synthesis) and repression of folate enzymes in mammalian tissues may indicate that the control of many of these enzymes is at the level of substrates or products which may act to stabilize their respective enzyme. Such enzymes would most likely be those which function in cycles to regenerate catalytic amounts of cofactors; e.g. enzymes 1-8 (Fig. 3). One such enzyme, FH2 reductase, is known to be stabilized by its substrates (71,72,38). Moreover, folate cofactors which are substrates of one enzyme may affect the activity of another folate enzyme, as in the case of 5-methyl FH4, a potent competitive inhibitor with respect to FH4 of serine transhydroxymethylase. Indeed, because

345

REGULATION OF DIHYDROFOLATE REDUCTASE HCOOH 4- FH 4 -I- ATP

®IL ADP 4-

[ I0- formyl FH 4 - -~- -'> formiminoglutamote I FH

~)

inosinte acid

I

®.'Jr .,o

(~) "NH 3

5- fofmlmino FH 4 + glutamate

serine ~- FH 4 ~

-- -- ->

formyl glyclnomide ribotide

(~) NADPfi I I NAOP glycine

"t"

purlnes

5,10-methylene FH4 - -

~

thyrnidylotl - --

-- - (~-- -).

methio4~ine - - - - >

-> DNA

ADPH Folote

FH

2

I 5-methylFH4 ]

proteins

FIG. 3 The interrelationships of folate derivatives, modified after Whiteley(9). The enzymes, indicated by numbers, are: 1, 10 formyl FH4 synthetase, 2,13yclohydrolase; 3, methylene FH4 dehydrogenase; 4,5-methyl FH4 reductas¢; 5, dihydrofolate reductase, 6, serine transhydroxymethylase; 7, formirninotetrahydrofolate cyclodeaminase; 8, glutamate formiminotransferase; 9, phosphoribosyl-aminoimidazolecarboxamide formyltransferase; 10, phosphofibosyl-glycineamide formyltransferase; 11, thymidylate synthetase; 12, 5-methyltetrahydrofolatehomocysteine transmethylase.

of the close interactions and interconversions of the various folate cofactors, specific feedback inhibition of enzymes 1-8 would be difficult to achieve because such inhibition would disrupt the cyclic reactions shown. Specific feedback inhibition might be more likely in the case of enzymes such as 9-12, where products are not cycled, but are further utilized in cellular metabolism. Finer control may be provided by intracellular localization of the various enzymes. For example, the rates of entry of substrates into and the egress of products from such intracellular compartments may control the reaction rates of some of these enzymes. Thymidyla.te synthetase has been found mainly in the nuclei of liver cells, while 12% of the FH2 reductase present was located in the mitochondria and 88% in the supernatant (74). As mentioned previously, serine transhydroxymethylase was found in the mitochondria to the largest extent (58%), the remainder being cytoplasmic (29, 3 3). Induction of folate enzymes involving synthesis of new enzyme protein has been demonstrated only as a result of viral infection, Phage infection of E. coliinduced thymidylate synthetase (75) and FH2 reductase

346

J. R. BERTINO AND B. L. HILLCOAT

(76,77) while FH2 reductase induction followed infection of mouse kidney cells with polyoma or SV40 virus (78). The mechanisms involved are not known. In phage infected E. coli, the intracellular concentration of N A D P H , a coenzyme known to stabilize FH2 reductase, increased in a parallel fashion with increase in enzyme activity(79); thus cofactor stabilization could play some role in this increase. The need for the FH2 reductase induced in phage infected E. coli has not been elucidated, especially since comparatively high levels of this enzyme are presentqn uninfected E. coli(79). In addition, the observed ability of phage mutants lacking thymidylate synthetase and/or FH2 reductase to grow relatively normally(80), makes extrapolation of these phenomenon to mammalian systems difficult. At present, the limited information available concerning the control of folate enzymes in mammalian systems suggests that cofactor induction may be an important mechanism of control. Further studies in this area are needed to confirm and define such a role. REFERENCES 1. L. JAENICKE, Vitamin and coenzyme function: vitamin B~ and folic acid, Ann. Rev. Biochem. 33, 287-312 (1961). 2. F. M. HUE~NEKENS, The role of dihydrofolic reductase in the metabolism of onecarbon units, Biochemistry 2, 15 ! - 159 (1963). 3. M. FRmDKIN, Enzymatic aspects of folic acid, Ann. Rev. Biochem. 32, 185-214 (1963). 4. J. C. RASINOWITZ, Folic acid, pp. 185-252 in The Enzymes Vol. 2, (P. D. BOYER, H. LARDY and K. MYRSACK, eds.) Academic Press, Inc., New York (1960). 5. J. C. RASINOWITZ and W. E. PRICER, JR., Formyltetrahydrofolate synthetase, I. Isolation and crystallization of the enzyme, J. BioL Chem. 237, 2898-2902 (1962). 6. H. R. WHITELEY, M. J. OSSORrq and F. M. HUENNEgENS, Purification and properties of the formate activating enzyme from Micrococcus aerogenes, J. Biol. Chem. 234, 1538-1543 (1959). 7. J. C. RASINOWITZ and W. E. PRICER, JR., A T P formation accompanying forminoglycine utilization, J. Am. Chem. Soc. 78, 1513-1517 (1956). 8. A. ALSRECHT and D. J. HUTCHINSON, Repression by adenine of the formyltetrahydrofolate synthetase in an antifolic-resistant mutant of Streptococcus faecalis, J. Bact. 87, 792-798 (1964). 9. H. R. WHITELEY, The distribution of the formate activating enzyme and other enzymes involving tetrahydrofolate in animal tissues, Comp. Biochem. Physiol. 1, 227-247 (1960). 10. J. R. BERTINO, B. SIMMONS and D. M. DONOrtUE, Levels of dihydrofolate reductase and the formate-activating enzyme activities in guinea pig tissues, Biochem. Pharmacol. 13, 225-233 (1964). 1 I. J. R. BERTINO, B. SIMMONS and D. M. DONOHUE, Purification and properties of the formate-activating enzyme from erythrocytes, J. Biol. Chem. 237, 1314-1318 (1962). 12. J. R. BERTINO, R. SILBER, M. FREEMAN, A. ALENTY, M. ABRECHT, B. W. GABmO and F. M HUENNEKENS, Studies of normal and leukemic leukocytes. IV. Tetrahydrofolatedependent enzyme systems and dihydrofolic reductase, J. Clin. lnvestig. 42, 899-1907 (1963). 13. J. R. BERTINO, The mechanism of action of the folate antagonists in man, Cancer Research 23, 1286-1306 (1963).

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